Method for detecting the degree of malignancy of each of the circulating tumor cells, and a kit for the same

ABSTRACT

The object of the present invention is to provide an evaluation method capable of accurately determining a metastasis of cancer, the stage of cancer progression, or the malignancy of cancer. 
     The object can be solved by a method for detecting the degree of malignancy of each of the circulating tumor cells, characterized by the following steps: (a) bringing an epithelial cell-binding component, which specifically binds to a marker molecule expressed on epithelial cells and is fluorescently-labeled or luminescent enzyme-labeled, and a mesenchymal cell-binding component, which specifically binds to a marker molecule expressed on mesenchymal cells and is fluorescently-labeled or luminescent enzyme-labeled, into contact with a sample that possibly contains circulating tumor cells, (b) detecting a fluorescence signal or a luminescence signal of the epithelial cell-binding component and a fluorescence signal or a luminescence signal of the mesenchymal cell-binding component of each of the cells, and (c) determining the degree of epithelial-mesenchymal transition of circulating tumor cells based on the signal amount of the epithelial cell-binding component (E) and the signal amount of the mesenchymal cell-binding component (M).

TECHNICAL FIELD

The present invention relates to a method for detecting the degree ofmalignancy of each of the circulating tumor cells, a kit for detectingthe degree of malignancy of the circulating tumor cells, and anapparatus for detecting the degree of malignancy of the circulatingtumor cells.

BACKGROUND ART

Recently, it has become important to select an effective anticancer drugfor each cancer patient by removing cancer cells from the cancerpatient, and diagnosing the cancer cells thoroughly, for example, amolecular biological analysis such as gene variation analysis. It isknown that extremely-low concentrated cancer cells can be detected inthe blood of the cancer patients. These cancer cells are referred to ascirculating tumor cell (hereinafter sometimes referred to as CTC). Thenumber of the detected CTC is correlated to a prognosis of the patient.Therefore, a measurement of CTC begins to be performed, so as toevaluate a stage of cancer progression.

When CTCs are detected in the peripheral blood of cancer patients,extremely-low concentrated CTCs which are one-billionth of highconcentrated blood cells, should be detected in the high concentratedblood cells. Thus, a count loss of CTCs or a contamination betweensamples of patients leads to a seriously wrong diagnosis. For example,the measurement of CTCs is carried out using CellSearch System (U.S.A).As disclosed in Patent reference 1, in the above technique, the cellsare subjected to nuclear staining and cytokeratin staining, and then thecells are reacted with CD326 antibody-immobilized magnetic beads. Theresulting cells are then allowed to float through a magnetic field.Fluorescent imaging of cells are obtained by scanning by laser beam andthen a human determines whether or not each of the cells is CTC, on thebasis of the fluorescent imaging. Further, there is a technique calledCTC-Chip as a method for measuring CTCs. As disclosed in Patentreference 2, in the above technique, a blood sample is passed through achip in which 80,000 micro posts are formed on a silicon wafer about thesize of a business card. Then, all of the 80,000 micro posts immobilizedwith anti-EpCAM antibody are image-recognized so as to identify andmeasure CTCs. Patent reference 3 discloses that CTCs detected by themethod disclosed in Patent reference 1, are recovered, and then genes ofthe recovered CTCs are analyzed by fluorescence in situ hybridization(hereinafter sometimes referred to as FISH). Patent reference 4 and 9discloses a flowcytometer using a disposable chip. A measurement withouta cross contamination of samples can be conducted with the aboveflowcytometer. Patent reference 5 discloses a conventional flowcytometer wherein a solution sending system including a flow cell isfixed. Patent reference 6 discloses that CTCs detected by the methoddisclosed in Patent reference 1, are recovered, and then an abnormalityof IGF-1R gene of CTCs is analyzed by FISH. Patent reference 8 disclosesthat CTCs are concentrated by a microfilter on the basis of cell size.Non-patent reference 1 discloses that a fluorescent protein i.e. GFP isexpressed in cancer cells by using a virus capable of infecting cancercells only, and the expressed cell is detected by a fluorescencemicroscope. Non-patent reference 1 discloses that when anepithelial-mesenchymal transition (hereinafter sometimes referred to asEMT) in cancer cells derived from an epithelial cell is induced, thecancer cells easily become detached and easily migrate to other regions.The EMT is proposed by Elizabeth Hay et al. in the early 1980's. In theEMT, the epithelial cell is morphologically altered to amesenchymal-like cell. Further, Non-patent reference 1 discloses that adegree of EMT is related to a metastasis of cancer. Non-patent reference2 discloses that an EMT is induced in CTC derived from epithelial cellalso. Non-patent reference 7 discloses findings on EMT of culture cellsby protein analysis using an electrophoresis. It is reported that aratio between an expression amount of cytokeratin and an expressionamount of vimentin is not altered, but constant during the cell cycle.The ratio between expressions is measured by data of electrophoresis,and therefore the ratio is the average of a number of cells.

In Non-patent reference 4, a method for separating CTCs derived fromprostate cancer from leucocytes and concentrating CTCs by means of CPTVacutainer tubes manufactured by Becton, Dickinson and Company isdisclosed. Non-patent reference 5 discloses a CYTOTRACK systemmanufactured by CYTOTRACK (U.S.A). The CYTOTRACK system does notcomprise a specific concentration step before detection. That is to say,cells containing CTCs are separately fixed on a disc after labellingwith a fluorescent antibody. Consequently, the CTCs on the disc aredetected by laser-scanning using a compact disc turntable system. InNon-patent reference 6, a method for magnetically-concentrating CTCsusing an anti-EpCAM antibody, and a method for detecting CTCs using theapparatus disclosed in Patent reference 4, are disclosed.

Further, as a reagent kit relevant to CTCs, there may be mentioned abasic research reagent kit for magnetically-concentrating CTCs fromperipheral blood (D326(EpCAM)Tumor Cell Enrichment and Detection Kit;Miltenyi Biotec, catalog number 130-090-500); a reagent kit formagnetically-concentrating epithelial cells (HUMAN EpCAM POSITIVESELECTION KIT; STEMCELL Technologies, catalog number 18356); a reagentkit for negatively-selecting CTCs from bloods by cross-linking othercells such as erythrocytes and leukocytes other than CTCs anddensity-gradiently centrifuging the cells (Tumor Cell EnrichmentCocktail; STEMCELL Technologies, catalog number 15167); a reagent kitfor concentrating CTCs by using antibody-immobilized magnetic beads andnegatively-selecting CTCs using magnets (Tumor Cell Enrichment Cocktail;STEMCELL Technologies, catalog number 14152).

CITATION LIST Patent Literature

[PATENT LITERATURE 1] US 2002/0172987

[PATENT LITERATURE 2] US 2007/0026469

[PATENT LITERATURE 3] Japanese Unexamined Patent Publication (Kokai) No.2007-178193

[PATENT LITERATURE 4] Japanese Unexamined Patent Publication (Kokai) No.2010-181349

[PATENT LITERATURE 5] Japanese Unexamined Patent Publication (Kokai) No.2006-29921

[PATENT LITERATURE 6] Japanese Translation Publication (Kohyo) No.2011-515109

[PATENT LITERATURE 7] WO 2011/086990

[PATENT LITERATURE 8] US 2006/0254972

[PATENT LITERATURE 9] Japanese Unexamined Patent Publication (Kokai) No.2010-14416

Non-Patent Literature

-   [NON-PATENT LITERATURE 1] The Journal of Clinical Investigation    (United States) 2009, vol. 119, p.3172-3181-   [NON-PATENT LITERATURE 2] Breast Cancer Research (Great Britain)    2011, vol. 13, R59-   [NON-PATENT LITERATURE 3] Journal of Mammary Gland Biology and    Neoplasia (Germany) 2010, vol. 15, p.117-134-   [NON-PATENT LITERATURE 4] Journal of Clinical Oncology (United    States) 2011, vol. 29 (suppl 7; abstr41)-   [NON-PATENT LITERATURE 5] CytoTrack Poster, 2009, A3.pdf, internet    (http://ing.dk/modules/fsArticle/download.php?fileid=550)-   [NON-PATENT LITERATURE 6] Cytometry Part A (United States) 2011,    vol. 79A, p.107-117-   [NON-PATENT LITERATURE 7] The Journal of Cell Biology (United    States) 1984, vol. 99, p.1424-1433

SUMMARY OF INVENTION Technical Problem

In the method for detecting and measuring CTCs, it is necessary todetect 1 cell/mL of CTC from 10⁹ cells/mL of other highly concentratedcells such as erythrocytes and leucocytes.

The first problem is related to a detection of CTCs wherein anepithelial-mesenchymal transition is induced, by using EpCAM, asmentioned below.

In the method disclosed in Patent reference 1, a protocol forconcentrating CTCs using an anti-EpCAM antibody is employed. However, inNon-Patent reference 2, it is reported that epithelial-mesenchymaltransition (EMT) is induced in the CTCs derived from epithelial cancercells. If the epithelial-mesenchymal transition is induced, it isconsidered that a single cancer cell which forms a mass of cancer canmigrate, and thus the cancer cell can easily metastasize. Further, anexpression of EpCAM on cancer cells is reduced by EMT. Therefore, in themethod for detecting CTCs by concentrating CTCs using an anti-EpCAMantibody, it is difficult to detect CTCs having a high malignancy and ahigh metastatic property, wherein EMT is progressively induced.

The second problem is related to an evaluation of the stages of cancerprogression according to a ratio of an induction ofepithelial-mesenchymal transition, as mentioned below.

It is determined that the more the number (concentration) of CTCincreases, the more cancer becomes advanced, on the basis of acorrelation between a detected number (concentration) of CTCs inperipheral blood and a patient's prognosis. However, Non-patentreference 2 discloses that a rate of EMT-induced CTC is high in patientswith metastatic cancer compared to patients with early-stage cancer.Thus, it is considered that the rate of EMT-induced CTC is related tothe patient's prognosis, in addition to the number (concentration) ofCTC. That is, it is important to analyze an EMT induction in each of theCTCs. As described in Non-Patent reference 2, in the method foranalyzing the EMT induction in each of the CTCs, cytokeratin (CK) andvimentin on CTC are doubly, fluorescently-stained. When it is confirmedfrom an image of fluorescence microscope that vimentin on CTC isfluorescently-stained in addition to cytokeratin (CK), it is determinedthat the EMT is induced. According to this report, EMT is induced in 70%or more of the CTCs of patients with early-stage cancer, and EMT isinduced in 100% of the CTCs of patients with metastatic cancer. However,as EMT is induced in 70% or more of the CTCs of cancer patients, andthus it is difficult to determine whether or not the cancer cellmetastasizes, on the basis of the rate of EMT-induced CTC. This isbecause the number of detected CTCs is generally about 10 in 7 mL ofperipheral blood, and thus about 10 of the number of detected CTCs perse contains an uncertainty of plus or minus 30%, taking into account astatistical error according to Poisson distribution. Therefore, it ismeaningless to discuss a variation from 70% (patients with early-stagecancer) to 100% (patients with metastatic cancer) of the rate of theCTCs with EMT, by using the number of detected CTCs with an uncertaintyof plus or minus 30%.

That is to say, EMT is induced in 70% of circulating tumor cells of thepatients with early-stage cancer, and thus it is very difficult toevaluate a stage of cancer progression or a malignancy of cancer fromthe rate of EMT induction in circulating tumor cells. Further, it isimpossible to adopt the method for evaluating EMT disclosed inNon-patent reference 7, as one for evaluating EMT of the circulatingtumor cells wherein the number thereof is low.

The object of the present invention is to provide an evaluation methodcapable of accurately determining metastasis of cancer, stage of cancerprogression, or malignancy of cancer.

Solution to Problem

The present inventors have conducted intensive studies into theevaluation method of circulating tumor cells. As a result, the presentinventors, surprisingly, found that the metastasis of cancer, stage ofcancer progression, or malignancy of cancer can be accurately diagnosedby measuring an amount of a marker (such as cytokeratin) expressed on aepithelial cell and an amount of a marker (such as vimentin) expressedon a mesenchymal cell, and expressing the same as an EMT index.

The present invention is based on the above findings.

Accordingly, the present invention relates to

[1] a method for detecting the degree of malignancy of each of thecirculating tumor cells, characterized by the following steps:(a) bringing an epithelial cell-binding component which specificallybinds to a marker molecule expressed on epithelial cells and isfluorescently-labeled or luminescent enzyme-labeled, and a mesenchymalcell-binding component which specifically binds to a marker moleculeexpressed on mesenchymal cells and is fluorescently-labeled orluminescent enzyme-labeled, into contact with a sample that possiblycontains circulating tumor cells,(b) detecting a fluorescence signal or a luminescence signal of theepithelial cell-binding component and a fluorescence signal or aluminescence signal of the mesenchymal cell-binding component of each ofthe cells, and(c) determining the degree of epithelial-mesenchymal transition ofcirculating tumor cells based on the signal amount of the epithelialcell-binding component (E) and the signal amount of the mesenchymalcell-binding component (M),[2] the method for detecting the degree of malignancy of each of thecirculating tumor cells of item [1], further comprising the step of (d)removing erythrocytes and/or leucocytes from the sample, before or afterstep (a),[3] the method for detecting the degree of malignancy of each of thecirculating tumor cells of item [1] or [2], wherein a leucocyte-bindingcomponent, which specifically binds to a marker molecule expressed onleucocytes and is fluorescently-labeled or luminescent enzyme-labeled,is brought into contact with a sample that possibly contains circulatingtumor cells in the step (a),[4] the method for detecting the degree of malignancy of each of thecirculating tumor cells of items [1] to [3], wherein the fluorescencesignal or the luminescence signal is detected by a flow cytometer or animage analyzer in the detection step (b),[5] the method for detecting the degree of malignancy of each of thecirculating tumor cells of items [1] to [4], wherein the degree ofepithelial-mesenchymal transition is represented by a formula selectedfrom a group consisting of formula (1)

P=M/(E+M)  (1),

formula (2)

P=E/(E+M)  (2),

formula (3)

P=Log [M/(E+M)]  (3), and

formula (4)

P=Log [E/(E+M)]  (4)

wherein P is the degree, E is the signal amount of the epithelialcell-binding component, and M is the signal amount of the mesenchymalcell-binding component, in step (c),[6] the method for detecting the degree of malignancy of each of thecirculating tumor cells of items [1] to [5], further comprising the stepof (e) setting a reference of state before an onset ofepithelial-mesenchymal transition (0) and a reference of state after atermination of epithelial-mesenchymal transition (1) by measuringparticles to which a fluorescence substance labeling the epithelialcell-binding component is bound, and particles to which a fluorescencesubstance labeling the mesenchymal cell-binding component is bound,[7] the method for detecting the degree of malignancy of each of thecirculating tumor cells of item [6], step (e) is carried out togetherwith steps (b) and (c),[8] the method for detecting the degree of malignancy of each of thecirculating tumor cells of items [1] to [7], wherein the epithelialcell-binding component is an antibody or aptamer specifically binding tocytokeratin, EpCAM, or E-cadherin, and the mesenchymal cell-bindingcomponent is an antibody or aptamer specifically binding to vimentin, orN-cadherin,[9] a kit for detecting the degree of malignancy of the circulatingtumor cells, comprising(a) an epithelial cell binding antibody which specifically binds to amarker molecule expressed on epithelial cells and isfluorescently-labeled or luminescent enzyme-labeled, and(b) a mesenchymal cell-binding antibody which specifically binds to amarker molecule expressed on mesenchymal cells and isfluorescently-labeled or luminescent enzyme-labeled,[10] the kit for detecting the degree of malignancy of the circulatingtumor cells of item [9], further comprising:(c) a leucocyte-binding antibody which specifically binds to a markermolecule expressed on leucocytes and is fluorescently-labeled orluminescent enzyme-labeled, and/or(d) particles to which a fluorescence substance or luminescent enzymelabeling the epithelial cell-binding antibody is bound, and (e)particles to which a fluorescence substance or luminescent enzymelabeling the mesenchymal cell-binding antibody is bound,[11] the kit for detecting the degree of malignancy of the circulatingtumor cells of item [9] or [10], wherein the epithelial cell-bindingantibody is an antibody specifically binding to cytokeratin, EpCAM, orE-cadherin, and the mesenchymal cell-binding antibody is an antibodyspecifically binding to vimentin, or N-cadherin,[12] a use for preparing a kit for detecting the degree of malignancy ofthe circulating tumor cells, of an epithelial cell binding antibodyspecifically binding to a marker molecule expressed on epithelial cells,and/or a mesenchymal cell-binding antibody specifically binding to amarker molecule expressed on mesenchymal cells,[13] the use for preparing a kit for detecting the degree of malignancyof the circulating tumor cells of item [12], wherein the epithelialcell-binding antibody is an antibody specifically binding tocytokeratin, EpCAM, or E-cadherin, and the mesenchymal cell-bindingantibody is an antibody specifically binding to vimentin, or N-cadherin[14] an apparatus for detecting the degree of malignancy of thecirculating tumor cells, characterized by comprising:(a) a means of receiving an amount of fluorescence signal or theluminescence signal of epithelial cell-binding component bound to eachof the circulating tumor cells and an amount of fluorescence signal orthe luminescence signal of mesenchymal cell-binding component bound toeach of circulating tumor cells, and(b) a means of determining the degree of epithelial-mesenchymaltransition of each of the circulating tumor cells based on the receivedamount of signal of the epithelial cell-binding component (E) and thereceived amount of signal of the mesenchymal cell-binding component (M),[15] the apparatus for detecting the degree of malignancy of thecirculating tumor cells of item [14], wherein the degree ofepithelial-mesenchymal transition in the means (b) is represented by aformula selected from a group consisting of formula (1)

P=M/(E+M)  (1),

formula (2)

P=E/(E+M)  (2),

formula (3)

P=Log [M/(E+M)]  (3), and

formula (4)

P=Log [E/(E+M)]  (4)

wherein P is the degree, E is a signal amount of the epithelialcell-binding component, and M is a signal amount of the mesenchymalcell-binding component,[16] the apparatus for detecting the degree of malignancy of thecirculating tumor cells of item [14] or [15], wherein the epithelialcell-binding component is an antibody specifically binding tocytokeratin, EpCAM, or E-cadherin, and the mesenchymal cell-bindingcomponent is an antibody specifically binding to vimentin, orN-cadherin,[17] a method for evaluating cells, characterized in that the amounts ofmultiple molecules expressed on each of the cells are analyzed in eachcell, and the characteristic feature of each cell is quantified andindicated by using the amounts of multiple molecules, in procedures formeasuring cells,[18] the method for evaluating cells of item [17], wherein the amountsof multiple molecules analyzed in each cell are an amount of markermolecules expressed on epithelial cells (E) and an amount of markermolecules expressed on mesenchymal cells (M), the characteristic featureof each cell is a degree of epithelial-mesenchymal transition which isobtained by a formula calculated from E and M,[19] an apparatus for evaluating cells, characterized in that theapparatus can measure cells, and the amounts of multiple moleculesexpressed on each of the cells are analyzed in each cell, and thecharacteristic feature of each cell is quantified and indicated by usingthe amounts of multiple molecules, in procedures for measuring cells,[20] the apparatus for evaluating cells of item [19], wherein theamounts of multiple molecules analyzed in each cell are an amount ofmarker molecules expressed on epithelial cells (E) and an amount ofmarker molecules expressed on mesenchymal cells (M), the characteristicfeature of each cell is a degree of epithelial-mesenchymal transitionobtained by a formula calculated from E and M.

Advantageous Effects of Invention

According to the method for detecting the degree of malignancy of eachof the circulating tumor cells in the present invention, the metastasisof cancer, stage of cancer progression, or malignancy of cancer can beaccurately diagnosed. Specifically, the detecting method of the presentinvention can be used in an occurrence prediction of metastasis afterextirpation of the cancer, or the monitoring of the stages of cancerprogression. Further, the present invention can be used in monitoringtherapeutic effects of anticancer drugs by measuring CTCs afteradministration of said anticancer drugs and evaluating the degree of EMTin the CTCs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing an analysis process for obtaining an EMT indexof the CTC from measured data. The scatter plot between a forwardscattered signal (FS) and a sideward scattering signal (SS) (a); thescatter plot between FL1 corresponding to FITC and FL4 corresponding toALEXA700 (b); the scatter plot between the spectrum of nuclear stainingby 7-AAD (FL3) and spectrum of ALEXA700 (FL4) (c); and the graph showingEMT indexes calculated using FL1 and FL2 (d), are shown.

FIG. 2 is a histogram showing the evaluated EMT index of the A549 cellline mixed into peripheral blood.

FIG. 3 is a histogram showing the relationship between EMT index andintensity of fluorescence signal (A), and histograms showing EMT indexesof 0% of the degree of EMT and 100% of the degree of EMT (B).

FIG. 4 is a set of graphs showing the evaluation results of detectionefficiencies of various cell lines mixed into peripheral blood of anormal human. The graphs show that a cell line of 0% of EpCAM expressioncan be detected, and the CTC wherein EMT is progressively induced canalso be detected.

FIG. 5 is a view showing an example of applying an evaluation result ofEMT index to a diagnosis of the possibility of metastasis of cancer.

DESCRIPTION OF EMBODIMENTS [1] Method for Detecting the Degree ofMalignancy of the Circulating Tumor Cells

The method for detecting the degree of malignancy of each of thecirculating tumor cells of the present invention comprises the steps of:(a) bringing an epithelial cell-binding component which specificallybinds to a marker molecule expressed on epithelial cells and isfluorescently-labeled or luminescent enzyme-labeled, and a mesenchymalcell-binding component which specifically binds to a marker moleculeexpressed on mesenchymal cells and is fluorescently-labeled orluminescent enzyme-labeled, into contact with a sample that may possiblycontain circulating tumor cells, (b) detecting a fluorescence signal ora luminescence signal of the epithelial cell-binding component and afluorescence signal or a luminescence signal of the mesenchymalcell-binding component of each of the cells, and (c) determining thedegree of epithelial-mesenchymal transition of circulating tumor cellsbased on the signal amount of the epithelial cell-binding component (E)and the signal amount of the mesenchymal cell-binding component (M).

The method for detecting the degree of malignancy of each of thecirculating tumor cells of the present invention further comprises thestep of (d) removing erythrocytes and/or leucocytes from the sample,before or after step (a).

The detecting method of the present invention can be used as a methodfor detecting (diagnosing) the metastasis of cancer, a method fordetecting (diagnosing) the stage of cancer progression, or a method formonitoring the therapeutic effects in cancer, in particular, formonitoring the therapeutic effects of an anticancer drug.

<<Contact Step (a)>>

In the contact step (a) in the detecting method of the presentinvention, a sample that may possibly contain circulating tumor cells isbrought into contact with an epithelial cell-binding component whichspecifically binds to a marker molecule expressed on epithelial cellsand is fluorescently-labeled or luminescent enzyme-labeled, and amesenchymal cell-binding component which specifically binds to a markermolecule expressed on mesenchymal cells and is fluorescently-labeled orluminescent enzyme-labeled.

(Epithelial Cell Marker)

The marker expressed on an epithelial cell (hereinafter, referred to asan epithelial cell marker) is not particularly limited as long as it wasexpressed on the circulating tumor cells, but includes, for example, asurface protein and a sugar chain of an epithelial cell. Specifically,cytokeratin, EpCAM, or E-cadherin can be used as the epithelial cellmarker, and in particular, cytokeratin is preferably. As thecytokeratin, at least one cytokeratin selected from a group consistingof CK1, CK4, CK5, CK8, CK10, CK14, CK15, CK16, CK18, and CK19 may beused, but the mixture of two or more thereof is preferable. Further,cytokeratin, EpCAM, and E-cadherin are labeled with the samefluorescence, and the total expression amount thereof can be used as theexpression amount of the marker expressed on an epithelial cell. Acombination of the above markers is not limited, but containscytokeratin, preferably.

(Epithelial Cell-Binding Component)

The epithelial cell-binding component which specifically binds to theepithelial cell marker is not limited, so long as it can be bound to theepithelial cell marker, but it includes, for example, antibody, antibodyfragment, antigen, DNA, RNA, receptor, ligand for receptor, enzyme,ligand for enzyme, enzyme analog, substrate for the enzyme to be basedon enzyme analog, lectin, or sugar chain. Specifically, when theepithelial cell marker is protein, antibody, antibody fragment, ligandfor receptor, ligand for enzyme, DNA (such as aptamer), or RNA may beused. Further, when the epithelial cell marker is sugar chain, antibody,antibody fragment, or lectin may be used.

(Marker Expressed on Mesenchymal Cell)

Marker expressed on mesenchymal cell (hereinafter, referred to as amesenchymal cell marker), is not particularly limited as long as it isexpressed on the circulating tumor cells, but includes, for example, asurface protein and a sugar chain of mesenchymal cell. Specifically,vimentin, Twist, and N-cadherin can be used as the mesenchymal cellmarker. Further, vimentin, Twist and N-cadherin are labeled by the samefluorescence, and the total expression amount thereof can be used as theexpression amount of the marker expressed on the mesenchymal cell. Acombination of the above markers is not limited, but contains vimentin,preferably.

(Mesenchymal Cell-Binding Component)

The mesenchymal cell-binding component which specifically binds to themesenchymal cell marker is not limited so long as it can be bound to themesenchymal cell marker, but includes, for example, antibody, antibodyfragment, antigen, DNA, RNA, receptor, ligand for receptor, enzyme,ligand for enzyme, enzyme analog, substrate for the enzyme to be basedon enzyme analog, lectin, or sugar chain. Specifically, when themesenchymal cell marker is protein, antibody, antibody fragment, ligandfor receptor, ligand for enzyme, DNA (such as aptamer), or RNA may beused. Further, when the mesenchymal cell marker is sugar chain,antibody, antibody fragment, or lectin may be used.

(Antibody or Antibody Fragment)

The antibody used as the epithelial cell-binding component or themesenchymal cell-binding component is not limited, but there may bementioned, for example, polyclonal antibody, monoclonal antibody,recombinant antibody, or antibody fragment having the antigen-bindingsite thereof. As the antibody fragment, there may be mentioned, forexample, F(ab′)₂, Fab′, Fab, Fv, or the like. The fragment of theantibody may be obtained by conventional methods, for example, bydigesting the antibody using a protease (such as pepsin, papain, or thelike) and purifying the resulting fragments by standard polypeptideisolation and purification methods.

(Fluorescent Label or Luminescent Enzyme Label)

The epithelial cell-binding component and mesenchymal cell-bindingcomponent are preferably fluorescently-labeled, or luminescentenzyme-labeled. A fluorescent substance used for fluorescent labeling isnot particularly limited, but there may be mentioned, for example, AMCA,Alexa Flour 350, Murina Blue, Cascade Blue, Cascade Yellow, PacificBlue, Alexa Flour 405, Alexa Flour 488, Qdot(R)605, FITC, PE/RD1,ECD/PE-TexasRed, PC5/SPRD/PE-Cy5, PC5.5/PE-Cy5.5, PE Alexa Flour 750,PC7/PE-Cy7, TRITC, Cy3, Texas Red, Alexa Flour 647, Alexa Flour 700,Cy5, Cy5.5, APC, APC7/APC-Cy7, APC Alexa Flour 750.

Further, the luminescent enzyme is not particularly limited, butincludes luciferase. An origin of the luciferase is not particularlylimited, but there may be mentioned a luciferase derived from firefly,or a luciferase derived from bacteria. The luminescent enzyme becomesluminous by reacting with a substrate specific to the luminescentenzyme. Thus, it may be preferably used in the analysis by the imageanalyzer, as mentioned below. As the substrate of luminescent enzyme, asubstrate specific to each luminescent enzyme may be used, and thus, thesubstrate can be appropriately selected. For example, if the luciferaseis used, a luciferin having substrate-specificity to each luciferase maybe used.

(Fluorescence or Luminescence Stain of Leucocyte)

The contact step (a) may comprise the procedure of bringing a samplethat may possibly contain circulating tumor cells into contact with theleucocyte-binding component which specifically binds to a markermolecule expressed on leucocytes and is fluorescently-labeled orluminescent enzyme-labeled.

By fluorescently-staining the marker expressed on leucocytes, thecirculating tumor cells can surely be separated from leucocytes using aflow cytometer or an image analyzer in the following detection step.Thus, an accuracy of detection of circulating tumor cells can beincreased.

A leucocyte marker is not particularly limited, so long as it isspecifically expressed on leucocytes, but includes, for example, CD45,CD2, CD3, CD4, CD5, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20,CD24, CD25, CD27, CD29, CD33, CD36, CD38, CD41, CD45, CD45RA, CD45RO,CD56, CD66b, CD66e, CD69, or CD124. In particular, CD45 is preferable.This is because most leucocytes have a CD45 marker. Further, theleucocyte-binding component which specifically binds to the leucocytes,is not particularly limited, but includes, for example, antibody,antibody fragment, antigen, DNA, RNA, receptor, ligand for receptor,enzyme, ligand for enzyme, enzyme analog, substrate for the enzyme to bebased on enzyme analog, lectin, or sugar chain. However, antibody orantibody fragment is preferable.

An antibody used as the leucocyte-binding component is not limited, butthe antibody described in the above item “(Antibody or antibodyfragment)” can be used. Further, a fluorescent substance or luminescentenzyme for labeling a leucocyte-binding component is not limited, butthe fluorescent substance or luminescent enzyme described in the aboveitem “(Fluorescent label or luminescent enzyme label)” can be used.

(Nuclear Stain)

Further, in the method for detecting the degree of malignancy ofcirculating tumor cells of the present invention, the nuclear portion ofthe cells in the sample may be stained. The circulating tumor cells canbe surely separated from the cell membrane fragments or the like bynuclear staining

(Sample)

A sample used in the method for detecting the degree of malignancy ofcirculating tumor cells of the present invention is not particularlylimited, so long as it is a sample obtained from a patient suspected ofhaving cancer. That is, the sample is not limited, so long as the samplemay possibly contain circulating tumor cells. Specifically, as theliquid sample that may possibly contain circulating tumor cells, forexample, there may be mentioned blood, urine, lymph fluid, tissue fluid,spinal fluid, ascites fluid, or pleural effusion. The peripheral bloodis preferable because it is easily collected by drawing blood.

Further, the cancer in the patient in which the sample is collected isan epithelial cancer. As for the epithelial cancer, there may bementioned bladder cancer, breast cancer, colorectal cancer, rectalcancer, kidney cancer, hepatic cancer, lung cancer, small cell lungcancer, esophageal cancer, gallbladder cancer, ovarian cancer,pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer,prostate cancer, squamous cancer, skin cancer, duodenal cancer, vaginalcancer, or brain cancer.

The term “circulating tumor cells” as used herein means extremely-lowconcentrated cancer cells detected in the bloods of cancer patients. Itis sometimes referred to as “circulating tumor cells in blood” or“circulating tumor cells in peripheral blood”.

The circulating tumor cells detected in the present invention are notparticularly limited. However, the circulating tumor cells are derivedfrom epithelial cancer, preferably. A circulating tumor cell capable ofinducing epithelial-mesenchymal transition (EMT) is preferable. There isa report that a half-life of the circulating tumor cells (cancer cells)is 1 to 2.4 hours. Many of the circulating tumor cells are killed byapoptosis. Thus, about several to several dozen of the circulating tumorcells merely exist in 10⁹/mL of the blood cells.

<<Detection Step (b)>>

In the detection step (b), a fluorescence signal or a luminescencesignal of epithelial cell-binding component or mesenchymal cell-bindingcomponent bound to circulating tumor cells is detected.

The fluorescence or luminescence signal bound to the epithelial cellmarker or mesenchymal cell marker is measured on each of the circulatingtumor cells. A cell having a certain threshold amount or more offluorescence or luminescence signal of the epithelial cell marker isdetected as the circulating tumor cells.

In order to detect the fluorescence or luminescence signal ofcirculating tumor cells, an apparatus capable of identifying a singlecell is required. For example, a flow cytometer, or an image analyzercan be used.

(Measurement Using Flow Cytometer)

Specifically, a flow cytometer using a disposable micro flow path chipis preferable, in view of the prevention of cross-contamination betweensamples. Further, a fluorescence signal of each of the low-concentratedCTCs can be detected with high-speed in the flow cytometer. Therefore,it is preferable to measure CTCs by using the above flow cytometerdisclosed in Patent references 4 and 9, wherein the measurement withouta cross-contamination of samples can be achieved through the disposablemicro flow path chip. In the flow cytometer, the fluorescently-stainedcells are measured. Further, the measured data is the fluorescent signalintensity of each of the cells, and therefore the signal intensity canbe used to quantify a degree of epithelial-mesenchymal transition ofeach of the cells. This feature is different from the followingmeasurement using the image analyzer.

(Measurement Using Image Analyzer)

In the image analyzer, the fluorescently-stained cells orself-luminously-stained cells are measured. That is, a distribution offluorescent intensity or luminescence intensity of cells is measured.Therefore, the measured data of the image analyzer is different fromthat of a flow cytometer. In particular, it is an image of lightintensity distribution showing several cells in a defined area. Thus, inorder to quantify the signal of each of the cells, a function fortracing an outline of the cell by image recognition is required.Specifically, an integral quantity of the fluorescent intensitydistribution of an epithelial cell-binding component within the outlineof the cell, and an integral quantity of fluorescent intensitydistribution of the mesenchymal cell-binding component within theoutline of the cell are calculated. Then, when the degree ofepithelial-mesenchymal transition is quantified in the next step, theabove integral quantities are essentially used.

<<Degree Determination Step (c)>>

In the degree determination step (c), the degree ofepithelial-mesenchymal transition is quantified by the signal amount ofthe epithelial cell-binding component (E) and the signal amount of themesenchymal cell-binding component (M).

The signal amount of the epithelial cell-binding component (E) means theexpression amount of the epithelial cell marker on a circulating tumorcell, and the signal amount of the mesenchymal cell-binding component(M) means the expression amount of the mesenchymal cell marker on acirculating tumor cell. Thus, the degree of epithelial-mesenchymaltransition is quantified using the expression amount of the epithelialcell marker and the expression amount of the mesenchymal cell marker.

(Procedure for Quantifying Degree of EMT)

The index of degree of EMT is not limited, as long as it is calculatedusing the signal amount (E) of the epithelial cell-binding component andthe signal amount (M) of the mesenchymal cell-binding component and aratio between the signal amount (M) and the signal amount (E) changes.In particular, an index wherein the more the signal amount (M) withrespect to the signal amount (E) increases, the more the degree of EMTincreases, is referred to as the “EMT index (P)” herein.

Therefore, as a preferable formula of the EMT index (P), for example,there may be mentioned the following:

Formula (1)

P=M/(E+M)  (1).

The above EMT index (P) is advantageous from the viewpoint of definingthe scope of the upper limit and lower limit from 0 to 1. On the otherhand, for example, if an EMT index (P) is “M/E”, an obtained value mayvary from zero to infinity. However, the closer the value of E is tozero, the more the EMT index (P) increases. That is, when the value of Eis close to a detection limit that is not accurate, the EMT index (P)increases. Thus, the EMT index (P) of “M/E” is unfavorable for an indexof diagnosis.

While, as a formula of the EMT index (P) showing the degree of EMA, thefollowing formula may be used.

Formula (2)

P=E/(E+M)  (2)

In this case, a value of 1 means that the EMT is not induced. The aboveEMT index (P) is advantageous from the viewpoint of defining the scopeof the upper limit and lower limit. In addition, the above EMT index (P)may be expressed as a logarithm thereof.

According to the after-mentioned Examples, the EMT index (P) will beconcretely explained.

P=[(Vimentin expression amount in CTC)/((CK expression amount inCTC)+(Vimentin expression amount in CTC))]

When the EMT is not induced, the above P is zero. When the Vimentinexpression amount increases, the above P is close to 1. Thus, the lowerlimit of zero and the upper limit of 1 are defined as values showing thedegree of the EMT. Therefore, the above P can be suitably used as anindex for a common diagnostic standard even in different apparatuses.

A concrete procedure for measuring the EMT index is as follows. CK onCTC is stained by FITC labeled anti CK antibody. Then a fluorescence ofFITC is measured at a wavelength range of 510 nm to 550 nm, and theresulting signal intensity is referred to as FL1. Further, Vimentin onCTC is stained by a PE labeled anti Vimentin antibody. Then afluorescence of said PE is measured at a wavelength range of 550 nm to600 nm, and the resulting signal intensity is referred to as FL2. TheEMT index (P) can be shown as a formula of [A(FL2/(FL1+FL2))+B] whereinA and B are apparatus constants. The A is the apparatus constant basedon variability between apparatuses of detection sensitivity offluorescence of FL1 and FL2, and the B is the apparatus constant definedby the zero level of the fluorescence signal. The constants are definedin such a way that P is 0 in the case of the Vimentin expression amountof 0, and P is 1 in the case of the CK expression amount of 0 and onlythe Vimentin expression. In connection to this, the type of fluorescencesubstance is not limited to the above types.

The apparatus constants of A and B can be determined as follows.

In order to obtain a standard linear of fluorescence signals of acondition “100% CK (FITC)” wherein cytokeratin only expresses andvimentin does not express, it is necessary to measure cellssingly-stained by FITC. However, the preparation of such cells inaddition to a sample to be tested on a patient requires time. Thus, astandard linear may be obtained by measuring particles wherein FITC isbound thereto using a flow cytometer. Similarly, a standard linear offluorescence signals of a condition “100% Vimentin (PE)” whereinvimentin only expresses and cytokeratin does not express, may beobtained by measuring particles wherein PE is bound thereto using theflow cytometer. When two kinds of such standard particles having asingle fluorescence spectrum and the CTCs of the sample aresimultaneously measured, a determination of reference as to the upperand lower limits of the EMT index, and the analysis of the EMT index ofCTCs of the sample can be conducted simultaneously, and whereby thevariability between apparatuses in the analysis of the EMT index can besurely corrected. The correction is important when a common thresholdvalue is applied to the results obtained from various apparatuses.

The line of “100% CK (FITC)” drawn in a scatter plot of FIG. 3(A) isdefined by a ratio between FL1 of the fluorescence spectrum of FITC andFL2, and data of particles or cells only having fluorescence of FITC aredistributed thereon. Similarly, data of particles or cells only havingfluorescence of PE are distributed on the line of “100% Vimentin (PE)”.The apparatus constants A and B are defined in such a way that the EMTindexes of two kinds of data, i.e. “100% CK (FITC)” and “100% Vimentin(PE)” are 0 and 1 respectively.

(Epithelial-Mesenchymal Transition)

When the epithelial-mesenchymal transition is induced, a cell-to-celladhesiveness is decreased by a transition from epithelial cell tomesenchymal cell. If the EMT is induced in cancer cells, the cancercells are removed from cancer tissue. Thus, it is considered that theinduced EMT leads to an increase of metastatic potential of cancercells, and therefore the malignancy of cancer cells progresses. Inconnection to this, if the signal amount (E) of the epithelialcell-binding component is major, the stage of cancer progression is low.If the signal amount (M) of the mesenchymal cell-binding component ismajor, the stage of cancer progression is high. The signal amount (E) ofthe epithelial cell-binding component means the expression amount of theepithelial cell marker, and the signal amount (M) of the mesenchymalcell-binding component means the expression amount of the mesenchymalcell marker.

Non-Patent reference 2 discloses that EMT is induced in 70% or more ofthe CTCs of patients with early-stage cancer, and EMT is induced in 100%of the CTCs of patients with metastatic cancer. However, Non-Patentreference 2 does not disclose that a stage of cancer progression in asingle cell varies by the expression amount of an epithelial cell markersuch as cytokeratin, and the expression amount of a mesenchymal cellmarker such as vimentin.

In the method for detecting the degree of malignancy of the circulatingtumor cells, the malignancy of circulating tumor cells, the predictionof metastasis of cancer, or the stage of cancer progression can bedetermined by using the EMT index, i.e. the ratio between the signalamount (E) of the epithelial cell-binding component and the signalamount (M) of the mesenchymal cell-binding component. Further, theoccurrence prediction of metastasis after the extirpation of cancer,monitoring the stages of cancer progression, or the monitoringtherapeutic effects of anticancer drugs can be performed by using theEMT index.

<<Removal Step (d)>>

The method for detecting the degree of malignancy of the circulatingtumor cells further comprises the step of (d) removing erythrocytesand/or leucocytes from the sample. The removal step (d) is not anessential step in the detection method of the present invention.However, as about several to several dozen of the circulating tumorcells merely exist in 10⁹/mL of blood cells, it is preferable to removeerythrocytes and/or leucocytes before or after the contact step (a). Inparticular, the removal step (d) is preferably carried out before thecontact step (a). The epithelial cell marker and the mesenchymal cellmarker on the CTCs are effectively stained by conducting the removalstep (d) before the contact step (a).

The protocol for removing erythrocytes is not particularly limited.However, ammonium chloride solution for removing erythrocytes, orcommercially available buffers for removing erythrocytes can be used.

Further, the protocol for removing leucocytes is not particularlylimited, but there may be mentioned the negative selection of leucocytesusing antibody-immobilized magnetic beads against a surface marker ofleucocytes and a magnet. This protocol is preferable because it is anEpCAM-independent, enrichment method.

<<Reference Setting Step (e)>>

The method for detecting the degree of malignancy of the circulatingtumor cells of the present invention further comprises the step of (e)setting a reference of state before an onset of epithelial-mesenchymaltransition (0) and a reference of state after a termination ofepithelial-mesenchymal transition (1) by measuring particles to which afluorescence substance labeling the epithelial cell-binding component isbound, and particles to which a fluorescence substance labeling themesenchymal cell-binding component is bound. The “particles to which afluorescence substance labeling the epithelial cell-binding component isbound” corresponds to the particles wherein FITC is bound thereto, andthe “particles to which a fluorescence substance labeling themesenchymal cell-binding component is bound” corresponds to theparticles wherein PE is bound thereto. However, the fluorescencesubstance is not limited to FITC or PE, but, for example, thefluorescence substances described in the item “(Fluorescent label orluminescent enzyme label)” can be used. Further, a particle is notparticularly limited as long as it can be used in a flow cytometer orimage analyzer, but includes cells or beads having a uniform size.

In the method for detecting the degree of malignancy of the circulatingtumor cells of the present invention, the reference setting step (e) maybe carried out independently. Further, it may be carried out togetherwith detection of particles in the detection step (b). Furthermore, itmay be carried out simultaneously in the degree determination step (c).However, if it is carried out in step (b) or (c), beads having uniformand smaller size than cells are preferably used, in order to distinguishbeads from the circulating tumor cells.

(Determination of Threshold Value of Cancer Malignancy)

FIG. 3 shows a method for determining threshold value, in order to usethe analysis results of malignancy of CTCs in addition to theconventional measured number of CTCs for diagnosis.

The detection and measurement of CTCs are carried out by the contactstep (a) and the detection step (b). Further, the EMT index iscalculated by the degree determination step (c), and thereby the degreeof epithelial-mesenchymal transition of each of the circulating tumorcells is determined. Then, “when the number of circulating tumor cellshaving a certain EMT index or more is more than the predetermined number(threshold value)” or “when an average of EMT index is more than thepredetermined value (threshold value)”, it can be determined that, forexample, “the patient has metastasis of cancer at the high frequency”.Further, when the number of CTCs or EMT index are not more than thepredetermined value (threshold value), it can be determined that, forexample, the patient has a low possibility of metastasis of cancer.Similarly, when the number of CTCs or EMT index are more than thepredetermined threshold value (cutoff value), it can be determined that“malignancy of cancer is high”, “stage of cancer is progressed”,“effects of anticancer drug is low”, or the like. When the number ofCTCs or EMT index are not more than the predetermined value (thresholdvalue: cutoff value) to the contrary, it can be determined that“malignancy of cancer is low”, “stage of cancer is not progressed”,“effects of anticancer drug is high”, or the like. The predeterminedvalue (threshold value: cutoff value) can be appropriately determinedaccording to each situation.

The predetermined value (threshold value: cutoff value) can bedetermined without limitation, as long as the metastasis of cancer, themalignancy of cancer, the stage of cancer progression, or the like isdiagnosed. That is to say, the various predetermined value (thresholdvalue: cutoff value) can be used according to each situation. However,the predetermined value (threshold value: cutoff value) is preferablydetermined by a controlled clinical trial.

[2] Kit for Detecting the Degree of Malignancy of the Circulating TumorCells

The kit for detecting the degree of malignancy of the circulating tumorcells of the present invention comprises (a) an epithelial cell-bindingantibody which specifically binds to a marker molecule expressed onepithelial cells and is fluorescently-labeled or luminescentenzyme-labeled, and (b) a mesenchymal cell-binding antibody whichspecifically binds to a marker molecule expressed on mesenchymal cellsand is fluorescently-labeled or luminescent enzyme-labeled. Further, thekit for detecting the degree of malignancy of the circulating tumorcells of the present invention further comprises (c) a leucocyte-bindingantibody which specifically binds to a marker molecule expressed onleucocytes and is fluorescently-labeled or luminescent enzyme-labeledand/or (d) particles to which a fluorescence substance or luminescentenzyme labeling the epithelial cell-binding antibody is bound, and (e)particles to which a fluorescence substance or luminescent enzymelabeling the mesenchymal cell-binding antibody is bound.

The detection kit of the present invention can be used for a method ofdetecting the degree of malignancy of each of the circulating tumorcells. Therefore, the detection kit of the present invention can also beused for the method for detecting (diagnosing) the metastasis of cancer,the method for detecting (diagnosing) the stage of cancer progression,or the method for monitoring the therapeutic effects in cancer, inparticular, for monitoring the therapeutic effects of an anticancerdrug. That is, the detection kit of the present invention may be used asa kit for the above methods.

<<Epithelial Cell-Binding Antibody (a)>>

The epithelial cell-binding antibody (a) can specifically bind to theepithelial cell marker described in the detection method of the presentinvention. As the epithelial cell marker, there may be mentioned onestated in the above item “(Epithelial cell marker)”, more preferablycytokeratin, EpCAM, or E-cadherin.

The antibody is not particularly limited, as long as it can bind to theepithelial cell marker, but includes polyclonal antibody, monoclonalantibody, recombinant antibody, or antibody fragment having theantigen-binding site thereof. As for the antibody fragment, there may bementioned, for example, F(ab′)₂, Fab′, Fab, Fv, or the like. Thefragment of the antibody may be obtained by conventional methods, forexample, by digesting the antibody using a protease (such as pepsin,papain, or the like) and purifying the resulting fragments by standardpolypeptide isolation and purification methods.

<<Mesenchymal Cell-Binding Antibody (b)>>

The mesenchymal cell-binding antibody (b) can specifically bind to themesenchymal cell marker described in the detection method of the presentinvention. As the mesenchymal cell marker, there may be mentioned onestated in the above item “(Mesenchymal cell marker)”, more preferablyvimentin, or N-cadherin.

The antibody is not particularly limited, as long as it can bind to themesenchymal cell marker, but includes polyclonal antibody, monoclonalantibody, recombinant antibody, or antibody fragment having theantigen-binding site thereof. As for the antibody fragment, there may bementioned, for example, F(ab′)₂, Fab′, Fab, Fv, or the like. Thefragment of the antibody may be obtained by conventional methods, forexample, by digesting the antibody using a protease (such as pepsin,papain, or the like) and purifying the resulting fragments by standardpolypeptide isolation and purification methods.

<<Leucocyte-Binding Antibody (c)>>

The leucocyte-binding antibody (c) can specifically bind to theleucocyte marker described in the detection method of the presentinvention. As the leucocyte marker, there may be mentioned markersstated in the above item “(Fluorescence or luminescence stain ofleucocyte)”, more preferably CD45.

The antibody is not particularly limited, as long as it can bind to theleucocytes marker, but includes polyclonal antibody, monoclonalantibody, recombinant antibody, or antibody fragment having theantigen-binding site thereof. As for the antibody fragment, there may bementioned, for example, F(ab′)₂, Fab′, Fab, Fv, or the like. Thefragment of the antibody may be obtained by conventional methods, forexample, by digesting the antibody using a protease (such as pepsin,papain, or the like) and purifying the resulting fragments by standardpolypeptide isolation and purification methods.

(Fluorescent Label or Luminescent Enzyme Label)

The epithelial cell-binding antibody (a), mesenchymal cell-bindingantibody (b), and leucocyte-binding antibody (c) arefluorescently-labeled or luminescent enzyme-labeled. Further, as thefluorescent substance or luminescent enzyme, one generally known in thisfield can be used without limitation. For example, the fluorescentsubstance or luminescent enzyme described in the above item“(Fluorescent label or luminescent enzyme label)” can be used.

(Particles to which Fluorescence Substance or Luminescent EnzymeLabeling the Epithelial Cell-Binding Antibody is Bound)

The kit of the present invention may comprise particles to which afluorescence substance or luminescent enzyme labeling the epithelialcell-binding antibody (a) is bound. The particles are used to determinea standard linear (100% CK (FITC)) of the circulating tumor cells whichmerely express an epithelial cell marker as described in FIG. 3.Therefore, when the epithelial cell-binding antibody (a) is labeled byFITC, the particles also are labeled by FITC.

(Particles to which Fluorescence Substance or Luminescent EnzymeLabeling the Mesenchymal Cell-Binding Antibody is Bound)

The kit of the present invention may comprise particles to which afluorescence substance or luminescent enzyme labeling the mesenchymalcell-binding antibody is bound. The particles are used to determine astandard linear (100% vimentin (PE)) of the circulating tumor cellswhich merely express a mesenchymal cell marker as described in FIG. 3.Therefore, when the mesenchymal cell-binding antibody (a) is labeled byPE, the particles are also labeled by PE.

The kit of the present invention may contain a manual that describes theuse of the particles to which the fluorescence substance or luminescentenzyme is bound, and/or the calculation of the EMT index.

The particles, to which the fluorescence substance or luminescent enzymeis bound, are not particularly limited. As for the particles, forexample, cells may be used, or polystyrene beads containing a singlefluorescence substance may be used. The diameter of the particle is notparticularly limited, but preferably a bit smaller than the diameter ofthe cell. The range of diameter is preferably 3 to 6 μm, and a uniformdiameter thereof is preferable.

(Use of Antibody or Particle for Preparing the Kit for Detecting theDegree of Malignancy of the Circulating Tumor Cells)

The epithelial cell-binding antibody (a), which specifically binds to amarker molecule expressed on epithelial cells, can be used to preparethe kit for detecting the degree of malignancy of the circulating tumorcells. Further, the mesenchymal cell-binding antibody (b), whichspecifically binds to a marker molecule expressed on mesenchymal cells,can be used to prepare the kit for detecting the degree of malignancy ofthe circulating tumor cells. The above-mentioned epithelial cell-bindingantibody (a) and the above-mentioned mesenchymal cell-binding antibody(b) can be used for preparation. Further, the leucocyte-binding antibody(c) can be used to prepare the kit for detecting the degree ofmalignancy of the circulating tumor cells.

The particles to which a fluorescence substance or luminescent enzymelabeling the epithelial cell-binding antibody is bound, and theparticles to which a fluorescence substance or luminescent enzymelabeling the mesenchymal cell-binding antibody is bound, can also beused to prepare the kit for detecting the degree of malignancy of thecirculating tumor cells.

[3] Apparatus for Detecting the Degree of Malignancy of the CirculatingTumor Cells

The apparatus for detecting the degree of malignancy of the circulatingtumor cells of the present invention comprises (a) a means of receivingan amount of fluorescence signal or the luminescence signal of theepithelial cell-binding component bound to each of the circulating tumorcells and an amount of fluorescence signal or the luminescence signal ofthe mesenchymal cell-binding component bound to each of circulatingtumor cells, and (b) a means of determining the degree ofepithelial-mesenchymal transition of each of the circulating tumor cellsbased on the received amount of signal of the epithelial cell-bindingcomponent (E) and the received amount of signal of the mesenchymalcell-binding component (M).

(Means of Receiving)

The detecting apparatus of the present invention can be used for themethod for detecting the degree of malignancy of each of the circulatingtumor cells, and has a means for receiving fluorescence signal amountsor the luminescence signal amounts of the epithelial cell-bindingcomponent, and fluorescence signal amounts or the luminescence signalamounts of the mesenchymal cell-binding component. Specifically, as forthe means, there may be mentioned a fluorescence detector or an emissiondetector.

(Means of Determining the Degree)

The means of determining the degree comprises the means capable ofcalculating the EMT index from the received signal amount of theepithelial cell-binding component (E) and the received signal amount ofthe mesenchymal cell-binding component (M). For example, the EMT indexcan be calculated by using a programmed computer, after measuring E andM.

A definitional equation of the EMT index is not limited so long as itmeans a ratio between the signal amount of the epithelial cell-bindingcomponent (E) and the signal amount of the mesenchymal cell-bindingcomponent (M), but includes formulas “M/(E+M)”, “E/(E+M)”, “Log[M/(E+M)]”, and “Log [E/(E+M)]”.

For example, the definitional identity can be expressed by formula (1)

P=M/(E+M)  (1),

formula (2)

P=E/(E+M)  (2),

formula (3)

P=Log [M/(E+M)]  (3), or

formula (4)

P=Log [E/(E+M)]  (4)

wherein P is degree, E is an amount of the signal of the epithelialcell-binding component, and M is an amount of the signal of themesenchymal cell-binding component.

In the detecting apparatus of the present invention, the means ofreceiving and the means of determining the degree can be conductedaccording to a computer program.

EXAMPLES

The present invention will now be further illustrated by, but is by nomeans limited to, the following Examples.

Example 1

In this Example, in order to establish the detection method of thepresent invention, A549 cell line derived from lung cancer was mixedwith the peripheral blood of the volunteer, as a substitute for CTC.Then, cytokeratin and vimentin were detected and the EMT index wascalculated.

Specifically, 45 mL of lysing buffer was added to 4 mL of peripheralblood in which A549 cells (100 cells) were mixed, and the whole wasmixed and allowed to stand on ice so as to lyse erythrocytes. 0.1 mL ofsolution wherein ImmunoTOKUI (On-chip Biotechnologies Co., Ltd) wasdiluted by a factor of 20 with PBS buffer containing 0.5% BSA and 2 mMEDTA (hereinafter referred to as T-buffer) was added thereto. Themixture was centrifuged, and the supernatant was aspirated so as toobtain a cell pellet. The resulting cell pellet was resuspended in 200μL of T-buffer, and 100 μL of Fc Blocking Reagent was added thereto. Thewhole was incubated at 4° C. for 15 minutes. After the incubation, 200μL of T-buffer was added to the whole so as to obtain a cell suspension.

The beads immobilized with a CD45 antibody for removing leucocytes wereprepared as follows. 400 μL of magnetic beads immobilized with a CD45antibody (Dynabeads, Life technologies) was poured into an Eppendorftube. 400 μL of T-buffer was added to the beads and the whole was gentlymixed by moving the tube up and down. Then a magnet was brought close tothe tube, and the tube was allowed to stand for about 1 minute so thatthe beads were completely trapped on the wall of the tube. Thesupernatant was gently removed, and 400 μL of T-buffer was added to thebeads. Then the mixture was stirred by a tapping to obtain a Dynabeadsmixture.

400 μL of the Dynabeads mixture was added to 500 μL of the cellsuspension, and the whole was rotated at 4° C. at 20 rpm for 30 minutes,so that the beads were reacted with cells. The tube was brought close tothe magnet, and the tube was allowed to stand for about 10 seconds sothat the beads were completely trapped on the wall of the tube. Thesupernatant was gently removed, and 500 μL of T-buffer was added to thebeads and the mixture was stirred. The mixture was centrifuged at 600 gfor 5 minutes and a supernatant was removed. 10 mL of 1×BD Lyse/Fixbuffer diluted with deionized water was added thereto. The mixture wasgently stirred by moving the tube up and down, and was allowed to standat room temperature for 20 minutes. After the fixation of the cells, themixture was centrifuged at 600 g for 5 minutes, and the cells werewashed by 2 mL of T-buffer again.

The cells were centrifuged at 600 g, for 5 minutes. Then the cells wereresuspended with 30 μL of antibody reaction liquid containing 44, ofantibodies i.e. Alexa700-labelled anti-CD45 antibody, PE-labelledanti-vimentin antibody, FITC-labelled anti-cytokeratin antibody, and 26μL of T-buffer, and were reacted with antibodies at 4° C. 1 mL ofT-buffer was added thereto, and then the cells were centrifuged at 600 gfor 5 minutes. The cells were resuspended in 200 μL of T-buffer so as toobtain a sample stained with Alexa700-labelled anti-CD45 antibody,PE-labelled anti-vimentin antibody, and FITC-labelled anti-cytokeratinantibody.

Next, a measurement without cross contamination between samples by theflow cytometry was conducted. In particular, the measurement was carriedout using a flow cytometer (FISHMAN-R, On-chip Biotechnologies Co., Ltd)wherein the disposable micro flow path chip made of acrylic resin isused as the flow cell. In this apparatus, four fluorescences can bedetected, and the detected signals are shown as FL1, FL2, FL3, and FL4,respectively. The wavelength regions of FL1, FL2, FL3, and FL4 are 510nm to 550 nm, 565 nm to 605 nm, 656 nm to 696 nm, and 700 nm to 850 nm,respectively.

The resulting sample was applied to the flow cytometer, andFITC-labelled anti-cytokeratin antibody, PE-labelled anti-vimentinantibody, nuclear staining agent i.e. 7-AAD, and Alexa700-labelledanti-CD45 antibody were detected by FL1, FL2, FL3, and FL4,respectively. The EMT index was calculated according to the followingprocedures.

In FIG. 1( a), the scatter plot between the forward scattered signal(FS) and the sideward scattering signal (SS) is shown. One dot in thescatter plot corresponds to one cell. Many of the cells are leucocyteswhich were supposed to be removed by the magnetic beads. It was foundthat an enormous number of leucocytes other than CTCs are containedtherein. Firstly, small cellular fragments were removed from the scatterplot. In FIG. 1( b), the scatter plot FL1 corresponding to FITC and FL4corresponding to ALEXA700 is shown. The CK positive cells labelled withFITC were selected from the scatter plot. In FIG. 1( c), the scatterplot showing FL3 corresponding to the spectrum of 7-AAD and FL4corresponding to the spectrum of ALEXA700 is described. Leucocytes werecompletely removed by selecting the CD45 negative cells from the scatterplot. FIG. 1( d) is a graph showing the EMT index of CK-positive CTCcells not containing leucocytes, which is calculated using FL1 and FL2.In the scatter plot, the EMT index on a longitudinal axis and the FS onan abscissa axis are plotted. It was found that the EMT indexes of A549cells were distributed on about 10%. The histogram of the EMT indexvalues of CTCs is shown in FIG. 2.

Example 2

In this example, a method for determining the apparatus constants A andB of the flow cytometer in the formula i.e. EMTindex=[A(FL2/(FL1+FL2))+B] used in Example 1 is explained.

In order to obtain a standard linear of fluorescence signals of a “100%CK (FITC)” condition wherein cytokeratin only expresses and vimentindoes not express, particles bound with FITC were measured by the flowcytometer. Further, in order to obtain a standard linear of fluorescencesignals of a “100% Vimentin (PE)” condition wherein vimentin onlyexpresses and cytokeratin does not express, particles bound with PE weremeasured by the flow cytometer.

In particular, the apparatus constants A and B are calculated asfollows. In order to obtain a standard linear of fluorescence signals ofa “100% CK (FITC)” condition wherein cytokeratin only expresses andvimentin does not express, the FITC-bound particles having a diameter of3 μm were measured by the flow cytometer so as to obtain a standardlinear. Further, in order to obtain a standard linear of fluorescencesignals of a “100% Vimentin (PE)” condition wherein vimentin onlyexpresses and cytokeratin does not express, PE-bound particles having adiameter of 3 μm were measured by the flow cytometer. The apparatusconstants A and B are defined in such a way that the values of EMTindexes of the above two types of particles are 0 and 1, respectively.This will be illustrated in the use of FIG. 2.

In FIG. 2(A), the relationship between an EMT value and a fluorescencesignal value is shown. The dashed line “100% CK (FITC)” described inFIG. 2 is a standard linear of 100% FITC(cytokeratin), which is obtainedby the measurement values of FITC-bound particles. The dashed line “100%Vimentin (PE)” described in FIG. 2 is a standard linear of 100%PE(vimentin), which is obtained by the measurement values of PE-boundparticles. If the cytokeratin or vimentin is singly expressed on a cell,the dot of the cell is on the standard linear of either of them.

The cytokeratin is labelled with FITC, and thus, the CTC data, whereinthe cytokeratin is only expressed thereon (100%) and the EMT is notinduced, is distributed on the standard linear of “100% CK (FITC)”corresponding to the FITC fluorescence spectrum. When a CTC is on theabove standard linear of “100% CK (FITC)”, an EMT index value thereof iszero (0). On the other hand, when the vimentin is only expressed thereon(100%), the CTC data is distributed on the standard linear of “100%Vimentin (PE)” corresponding to the PE fluorescence spectrum. When a CTCis on the standard linear of “100% Vimentin (PE)”, an EMT index valuethereof is 1.

FIG. 3(B) is a set of histograms showing the data of particles boundwith PE and the data of particles bound with FITC. The apparatusconstants A and B are initially set in such a way that the modes thereofare 1 and 0 (zero), respectively. Therefore, the distributions of EMTindexes between various apparatuses can be compared. Generally, raw dataof fluorescence signal intensity in flow cytometry may not be comparedbetween various apparatuses with differences or the like. However, anindex for medical diagnosis which can be used for comparison betweenvarious apparatuses is required. The above EMT index satisfies thisrequest.

Example 3

In this Example, recovery rates in the detection method of the presentinvention were examined by using A549 cells or cancer cells other thanA549 cells.

The procedures described in Example 1 were repeated using A549 cells.Further, the procedures described in Example 1 were repeated except thatKATO-III cells, PC-9 cells, or PC-14 cells were used instead of A549cells. Then, EMT indexes thereof were calculated, and further detectionrates of these cells were calculated simultaneously.

As shown in FIG. 4, the detection rates of KATO-III cells, A549 cells,PC-9 cells, and PC-14 cells were approximately 100%, 89%, 75%, and 100%,respectively.

The detection rate of PC-14 cells in which EpCAM was not expressed, was100%. Therefore, it was revealed that the detection method of thepresent invention can effectively detect CTCs of metastatic cancer inwhich the EMT was induced.

Finally, a characteristic feature of the present invention is shown fromthe other point of view.

In the apparatus measuring the fluorescence signal intensity of each ofthe cells, an analog value showing a cell characteristic feature (degreeof EMT of CTC in the present invention), which is defined by multiplesignal intensities and an apparatus constant, was calculated, and anevaluation of the analog value corrected by the apparatus constant wasachieved. That is to say, the measurement data of each of the cellsbetween various apparatuses can be compared. In other words, the analoginformation of each of the cells can be used on medical diagnosis in themethod of the present invention. In the conventional flow cytometer,numbers of cells detected in a certain range of signal intensity areused as information for diagnosis. The conventional information is notthe analog information of each of the cells, but merely the numbers ofdetected cells. That is, it is impossible to express the analog valueshowing a characteristic feature of each of the cells in theconventional flow cytometer. In other words, according to the presentinvention, the analog value of each of the cells present in the samplesof the patients can be used for medical diagnosis.

INDUSTRIAL APPLICABILITY

The present invention has advantages in that (1) CTCs are detected byonly a blood drawing from patients so that cancer can be found. Further,the present invention has advantages in that (2) in the patients whereinthe cancer tissue was removed by surgery, a recurrence can be foundearly by only a blood drawing after surgery.

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are possible without departing from the scope of theappended claims.

1. A method for detecting the degree of malignancy of each of thecirculating tumor cells, characterized by the following steps: (a)bringing an epithelial cell-binding component which specifically bindsto a marker molecule expressed on epithelial cells and isfluorescently-labeled or luminescent enzyme-labeled, and a mesenchymalcell-binding component which specifically binds to a marker moleculeexpressed on mesenchymal cells and is fluorescently-labeled orluminescent enzyme-labeled, into contact with a sample that possiblycontains circulating tumor cells, (b) detecting a fluorescence signal ora luminescence signal of the epithelial cell-binding component and afluorescence signal or a luminescence signal of the mesenchymalcell-binding component of each of the cells, and (c) determining thedegree of epithelial-mesenchymal transition of circulating tumor cellsbased on the signal amount of the epithelial cell-binding component (E)and the signal amount of the mesenchymal cell-binding component (M). 2.The method for detecting the degree of malignancy of each of thecirculating tumor cells according to claim 1, further comprising thestep of (d) removing erythrocytes and/or leucocytes from the sample,before or after step (a).
 3. The method for detecting the degree ofmalignancy of each of the circulating tumor cells according to claim 1,wherein a leucocyte-binding component, which specifically binds to amarker molecule expressed on leucocytes and is fluorescently-labeled orluminescent enzyme-labeled, is brought into contact with a sample thatpossibly contains circulating tumor cells in the step (a).
 4. The methodfor detecting the degree of malignancy of each of the circulating tumorcells according to claim 1, wherein the fluorescence signal or theluminescence signal is detected by a flow cytometer or an image analyzerin the detection step (b).
 5. The method for detecting the degree ofmalignancy of each of the circulating tumor cells according to claim 1,wherein the degree of epithelial-mesenchymal transition is representedby a formula selected from a group consisting of formula (1)P=M/(E+M)  (1),formula (2)P=E/(E+M)  (2),formula (3)P=Log [M/(E+M)]  (3), andformula (4)P=Log [E/(E+M)]  (4) wherein P is the degree, E is the signal amount ofthe epithelial cell-binding component, and M is the signal amount of themesenchymal cell-binding component, in step (c).
 6. The method fordetecting the degree of malignancy of each of the circulating tumorcells according to claim 1, further comprising the step of (e) setting areference of state before an onset of epithelial-mesenchymal transition(0) and a reference of state after a termination ofepithelial-mesenchymal transition (1) by measuring particles to which afluorescence substance labeling the epithelial cell-binding component isbound, and particles to which a fluorescence substance labeling themesenchymal cell-binding component is bound.
 7. The method for detectingthe degree of malignancy of each of the circulating tumor cellsaccording to claim 6, step (e) is carried out together with steps (b)and (c).
 8. The method for detecting the degree of malignancy of each ofthe circulating tumor cells according to claim 1, wherein the epithelialcell-binding component is an antibody or aptamer specifically binding tocytokeratin, EpCAM, or E-cadherin, and the mesenchymal cell-bindingcomponent is an antibody or aptamer specifically binding to vimentin, orN-cadherin.
 9. A kit for detecting the degree of malignancy of thecirculating tumor cells, comprising (a) an epithelial cell bindingantibody which specifically binds to a marker molecule expressed onepithelial cells and is fluorescently-labeled or luminescentenzyme-labeled, and (b) a mesenchymal cell-binding antibody whichspecifically binds to a marker molecule expressed on mesenchymal cellsand is fluorescently-labeled or luminescent enzyme-labeled.
 10. The kitfor detecting the degree of malignancy of the circulating tumor cellsaccording to claim 9, further comprising: (c) a leucocyte-bindingantibody which specifically binds to a marker molecule expressed onleucocytes and is fluorescently-labeled or luminescent enzyme-labeled,and/or (d) particles to which a fluorescence substance or luminescentenzyme labeling the epithelial cell-binding antibody is bound, and (e)particles to which a fluorescence substance or luminescent enzymelabeling the mesenchymal cell-binding antibody is bound.
 11. The kit fordetecting the degree of malignancy of the circulating tumor cellsaccording to claim 9, wherein the epithelial cell-binding antibody is anantibody specifically binding to cytokeratin, EpCAM, or E-cadherin, andthe mesenchymal cell-binding antibody is an antibody specificallybinding to vimentin, or N-cadherin.
 12. A use for preparing a kit fordetecting the degree of malignancy of the circulating tumor cells, of anepithelial cell binding antibody specifically binding to a markermolecule expressed on epithelial cells, and/or a mesenchymalcell-binding antibody specifically binding to a marker moleculeexpressed on mesenchymal cells.
 13. The use for preparing a kit fordetecting the degree of malignancy of the circulating tumor cellsaccording to claim 12, wherein the epithelial cell-binding antibody isan antibody specifically binding to cytokeratin, EpCAM, or E-cadherin,and the mesenchymal cell-binding antibody is an antibody specificallybinding to vimentin, or N-cadherin.
 14. An apparatus for detecting thedegree of malignancy of the circulating tumor cells, characterized bycomprising: (a) a means of receiving an amount of fluorescence signal orthe luminescence signal of epithelial cell-binding component bound toeach of the circulating tumor cells and an amount of fluorescence signalor the luminescence signal of mesenchymal cell-binding component boundto each of circulating tumor cells, and (b) a means of determining thedegree of epithelial-mesenchymal transition of each of the circulatingtumor cells based on the received amount of signal of the epithelialcell-binding component (E) and the received amount of signal of themesenchymal cell-binding component (M).
 15. The apparatus for detectingthe degree of malignancy of the circulating tumor cells according toclaim 14, wherein the degree of epithelial-mesenchymal transition in themeans (b) is represented by a formula selected from a group consistingof formula (1)P=M/(E+M)  (1),formula (2)P=E/(E+M)  (2),formula (3)P=Log [M/(E+M)]  (3), andformula (4)P=Log [E/(E+M)]  (4) wherein P is the degree, E is a signal amount ofthe epithelial cell-binding component, and M is a signal amount of themesenchymal cell-binding component.
 16. The apparatus for detecting thedegree of malignancy of the circulating tumor cells according to claim14, wherein the epithelial cell-binding component is an antibodyspecifically binding to cytokeratin, EpCAM, or E-cadherin, and themesenchymal cell-binding component is an antibody specifically bindingto vimentin, or N-cadherin.
 17. A method for evaluating cells,characterized in that the amounts of multiple molecules expressed oneach of the cells are analyzed in each cell, and the characteristicfeature of each cell is quantified and indicated by using the amounts ofmultiple molecules, in procedures for measuring cells.
 18. The methodfor evaluating cells according to claim 17, wherein the amounts ofmultiple molecules analyzed in each cell are an amount of markermolecules expressed on epithelial cells (E) and an amount of markermolecules expressed on mesenchymal cells (M), the characteristic featureof each cell is a degree of epithelial-mesenchymal transition which isobtained by a formula calculated from E and M.
 19. An apparatus forevaluating cells, characterized in that the apparatus can measure cells,and the amounts of multiple molecules expressed on each of the cells areanalyzed in each cell, and the characteristic feature of each cell isquantified and indicated by using the amounts of multiple molecules, inprocedures for measuring cells.
 20. The apparatus for evaluating cellsaccording to claim 19, wherein the amounts of multiple moleculesanalyzed in each cell are an amount of marker molecules expressed onepithelial cells (E) and an amount of marker molecules expressed onmesenchymal cells (M), the characteristic feature of each cell is adegree of epithelial-mesenchymal transition obtained by a formulacalculated from E and M.